It is known that genetic codes contained in genomes of organism individuals belonging to the same species are not identical each other, and there are differences in base sequences called polymorphisms. Ones in which one to tens of base(s) is (are) deleted or inserted, ones in which a specific base sequence is duplicated and the like are known as polymorphisms. One in which one base is replaced by another base is called a single nucleotide polymorphism (SNP).
It is said that single nucleotide polymorphisms exist at a rate of about one per hundreds to one thousand bases. Accordingly, the number of SNPs present on a human genome is estimated to be three to ten million. Attentions are paid to SNPs as indexes for searching for genes related to diseases, or for having information about differences in susceptibilities to diseases or sensitivities to drugs (actions or side effects). Methods for detecting SNPs are under study.
Conventional means for detecting SNPs are generally classified into ones based on hybridization, ones based on primer extension and ones utilizing substrate specificities of enzymes.
The presence of a base substitution is detected by means of hybridization of a probe to a nucleic acid sample in a hybridization method. According to the method, it is necessary to determine a probe and hybridization conditions so that hybridization is influenced by a difference in one base. Therefore, it is difficult to establish a highly reproducible detection system.
A method for detecting a mutation using a cycle probe reaction as described in U.S. Pat. No. 5,660,988 is exemplified. A nucleic acid probe having a readily cleavable binding is hybridized to a nucleic acid molecule of interest in the method. If the nucleic acid molecule of interest does not have a base substitution, the probe is cleaved, whereas if the nucleic acid molecule has a base substitution, the probe is not cleaved. A base substitution is then detected by detecting and quantifying the degree of generation of a fragment released from the cleaved probe. However, if a trace amount of a target nucleic acid is to be detected according to this method, there may be a considerable time lag until reaching a level at which one can detect a cleavage product from the probe because the amount of the cleavage product is small.
A method for detecting a mutation using the TaqMan method as described in U.S. Pat. Nos. 5,210,015 and 5,487,972 exemplifies another method. A TaqMan probe to which a fluorescent dye and a quencher are attached is used in this method. Two probes (one containing a base substitution and the other containing no base substitution) are used as the TaqMan probes. The probe is hybridized to a nucleic acid molecule of interest, and a primer is extended from the upstream. The probe is cleaved due to a 5′→3′ exonuclease activity of a DNA polymerase only if the nucleic acid molecule of interest does not contain a base substitution. A base substitution is then detected by detecting emitted fluorescence. However, the method has problems because the method requires a polymerase having a 5′→3′ exonuclease activity, a PCR using a labeled nucleotide blocked at the 3′-terminus and a strict temperature adjustment, and it requires a long time for detection.
Methods in which an enzyme is utilized include methods in which a DNA polymerase is used. Such methods are further classified into three groups as follows: (1) methods in which a base substitution is detected based on the presence of a primer extension reaction using a primer of which the 3′-terminus anneals to a base portion for which a base substitution is to be detected as described in U.S. Pat. No. 5,137,806; (2) methods in which a base substitution is detected based on the presence of a primer extension reaction using a primer in which the base substitution site to be detected is located at the second nucleotide from the 3′-terminus as described in WO 01/42498; and (3) method in which the presence of a mutation at the site of interest and the base at the site are determined by distinguishing a base incorporated into a primer using a primer of which the 3′-terminus anneals to a base 3′ adjacent to the base for which a base substitution is to be detected.
Methods in which a DNA ligase is used are known. According to the method, a base substitution is detected based on the presence of ligation of a probe to an adjacent probe. The terminal portion of the probe corresponds to the base portion for which a base substitution is to be detected.
A method in which a DNA polymerase or a DNA ligase is used may not be able to exactly detect a mismatch between a primer (or a probe) and a target nucleic acid due to a base substitution. Specifically, such an enzyme may initiate an enzymatic reaction even if the primer or the probe has a mismatch, providing erroneous results.
Because of a possible false positive due to an erroneous annealing between a target nucleic acid and a primer or an error made by a ligase or a polymerase to be used, it is necessary to control the reaction conditions (in particular, the reaction temperature) and the like very strictly, and there is a problem concerning the reproducibility.
Lastly, methods in which an enzyme having an activity of recognizing and cleaving a specific structure in a double-stranded nucleic acid is utilized such as the invader method as described in U.S. Pat. No. 5,846,717 are included. A cleavase is known as such an enzyme. It is possible to detect a base substitution by examining cleavage of a probe. The probe is designed such that it forms a structure recognized by the enzyme if a base substitution is present (or absent). However, such a method in which an enzyme having an activity of recognizing and cleaving a specific structure in a double-stranded nucleic acid is used has a problem concerning its sensitivity. Specifically, a signal sufficient for detection of a base substitution cannot be obtained from a trace amount of a nucleic acid sample since one signal is generated from one target nucleic acid molecule according to the method. It is naturally possible to enhance the signal by repeating the probe cleavage reaction, although it is necessary to amplify a target nucleic acid beforehand in order to obtain an intense signal. Thus, if a trace amount of a target nucleic acid is to be detected according to this method, there may be a considerable time lag until reaching a level at which one can detect a cleavage product from the probe because the amount of the cleavage product is small.
Since the methods have several problems as described above, a method that can be used to exactly detect a base substitution has been desired.